WO2023287212A1 - Épitope d'antigène de surface de lymphocyte t régulateur, et anticorps se liant de manière spécifique à celui-ci - Google Patents

Épitope d'antigène de surface de lymphocyte t régulateur, et anticorps se liant de manière spécifique à celui-ci Download PDF

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WO2023287212A1
WO2023287212A1 PCT/KR2022/010262 KR2022010262W WO2023287212A1 WO 2023287212 A1 WO2023287212 A1 WO 2023287212A1 KR 2022010262 W KR2022010262 W KR 2022010262W WO 2023287212 A1 WO2023287212 A1 WO 2023287212A1
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disease
antibody
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amino acid
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김정호
김범석
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주식회사 굳티셀
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Priority to CN202280050296.0A priority Critical patent/CN117716238A/zh
Priority to EP22842474.3A priority patent/EP4375670A1/fr
Publication of WO2023287212A1 publication Critical patent/WO2023287212A1/fr
Priority to US18/413,322 priority patent/US20240141036A1/en

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    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
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Definitions

  • the present invention relates to an epitope of Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein, which is an antigen present on the surface of regulatory T cells, and an antibody or antigen-binding fragment specifically binding thereto.
  • Lrig-1 leucine-rich and immunoglobulin-like domains 1
  • the most important characteristic of any normal organism is the ability to recognize and eliminate non-self antigens while not reacting detrimentally to antigenic substances that make up the self. .
  • Such unresponsiveness of the living body to self-antigens is referred to as immunologic unresponsiveness or tolerance.
  • Self-tolerance occurs by eliminating lymphocytes that may have specific receptors for self-antigens, or by inactivating self-reactive functions after encountering self-antigens. When a problem arises in inducing or maintaining self-tolerance, an immune response against self-antigens occurs, and a disease caused by this is called an autoimmune disease.
  • the regulatory T cells play an important role in naturally preventing the occurrence of excessive inflammation and immune response, but when autoimmune diseases and chronic inflammatory diseases occur, the function and number of regulatory T cells are significantly reduced. reported Therefore, in the case of patients with immune and inflammatory diseases, it is important that regulatory T cells are produced at normal levels, and this can be one of the treatments for these diseases.
  • CDRs complementarity determining regions
  • the CDR mainly serves to bind to the epitope of the antigen.
  • the CDRs of each chain are typically referred to sequentially starting from the N-terminus as CDR1, CDR2, and CDR3, and are also identified by the chain in which the particular CDR is located.
  • One object of the present invention is to provide an epitope of Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present on the surface of regulatory T cells (Treg cells).
  • Another object of the present invention is to provide an antibody or antigen-binding fragment capable of specifically binding to the epitope.
  • Another object of the present invention is a nucleic acid molecule encoding the epitope of the present invention; an expression vector into which the nucleic acid molecule is inserted; and a host cell line transfected with the expression vector.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating various diseases comprising an antibody or antigen-binding fragment capable of specifically binding to the epitope as an active ingredient.
  • Another object of the present invention is to provide an antibody-drug conjugate (ADC) in which the antibody according to the present invention is combined with a drug for preventing or treating various diseases.
  • ADC antibody-drug conjugate
  • Another object of the present invention is to treat various diseases including antibody-drug conjugates as an active ingredient, for example, immune-related diseases;
  • various diseases including antibody-drug conjugates as an active ingredient, for example, immune-related diseases;
  • Another object of the present invention is an antibody or antigen-binding fragment capable of specifically binding to the epitope; and methods for preventing or treating various diseases using antibody-drug conjugates.
  • the present invention relates to an epitope of Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein or an antibody or antigen-binding fragment that specifically binds to the epitope.
  • Lrig-1 leucine-rich and immunoglobulin-like domains 1
  • the "epitope” refers to a sequence of amino acids within an antigen in which amino acids (or a subset thereof) within the sequence are specifically recognized by an antibody or binding fragment, e.g., an antibody or binding fragment described herein. do.
  • An epitope may include one or more antigenic determinants. For example, antibodies raised against an isolated peptide corresponding to a conformational epitope recognize some or all of that epitope sequence.
  • the "Lrig-1 protein” is a transmembrane protein present on the surface of regulatory T cells, and includes an extracellular or lumenal leucine-rich repeat (LRR) and three immune-like domains. (immunoglobulin-like domains), transmembrane sequences, and cytoplasmic tails.
  • the LRIG gene family includes LRIG1, LRIG2 and LRIG3, and the amino acids constituting each family are highly conserved.
  • the LRIG1 gene is highly expressed in normal skin, and can regulate the proliferation of epithelial stem cells by expressing it in basal and hair follicle cells.
  • the Lrig-1 protein may be a protein derived from mammals or mice, but is not limited thereto.
  • the Lrig-1 protein may be Lrig-1 protein derived from mammals, such as humans, primates such as monkeys, and rodents such as mice and rats.
  • the Lrig-1 protein may be human-derived Lrig-1 protein represented by SEQ ID NO: 1, which may be encoded by the nucleic acid sequence represented by SEQ ID NO: 2, but is not limited thereto.
  • the Lrig-1 protein may be mouse-derived Lrig-1 protein represented by SEQ ID NO: 3, which may be encoded by the nucleic acid sequence represented by SEQ ID NO: 4, but is not limited thereto.
  • the Lrig-1 protein may be an extracellular domain of the Lrig-1 protein, but is not limited thereto.
  • the Lrig-1 extracellular domain of the present invention may be an extracellular domain of the Lrig-1 protein derived from mammals, such as humans, primates such as monkeys, and rodents such as mice and rats.
  • the extracellular protein of the Lrig-1 protein may be an extracellular domain of the Lrig-1 protein derived from human or mouse, but is not limited thereto.
  • the extracellular domain of the Lrig-1 protein may be represented by SEQ ID NO: 5 corresponding to the 35th to 794th amino acid sequence of the human-derived Lrig-1 protein, but is not limited thereto.
  • the extracellular domain of the Lrig-1 protein may be represented by SEQ ID NO: 6 corresponding to the 35th to 794th amino acid sequence of the mouse-derived Lrig-1 protein, but is not limited thereto.
  • an epitope of the Lrig-1 protein comprising at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences represented by SEQ ID NOs: 35 to 45 is provided.
  • the epitope of the Lirg-1 protein may be an epitope comprising at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences represented by SEQ ID NOs: 35 to 40, but is not limited thereto not.
  • the epitope of the Lrig-1 protein may be an epitope comprising at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences represented by SEQ ID NO: 41 to SEQ ID NO: 45. It is not limited.
  • the epitope of the Lrig-1 protein is located at positions 62 to 101, or 88 to 92, or 90 to 101, or 111 to 134, or 452 of the Lrig-1 protein. It may be a site corresponding to the 476th, or 472nd to 480th, or 542nd to 553rd base sequence.
  • the epitope of the Lrig-1 protein may be a region corresponding to the 95th to 101st base sequence or the 472nd to 476th base sequence of the Lrig-1 protein.
  • the epitope of the present invention may be a conformational epitope.
  • the "conformational epitope” of the present invention is composed of a discontinuous amino acid sequence. These conformational epitopes react with the three-dimensional structure of the antibody antigen-binding site.
  • nucleic acid molecule encoding the epitope provided in the present invention is provided.
  • Nucleic acid molecules of the present invention include all nucleic acid molecules in which the amino acid sequence of a polypeptide provided in the present invention is translated into a polynucleotide sequence as known to those skilled in the art. Therefore, various polynucleotide sequences by ORF (open reading frame) can be prepared, and all of them are also included in the nucleic acid molecule of the present invention.
  • ORF open reading frame
  • an expression vector into which the isolated nucleic acid molecule provided in the present invention is inserted is provided.
  • the "vector” is a nucleic acid molecule capable of transporting another nucleic acid to which a nucleic acid molecule is linked.
  • a vector is a “plasmid,” which refers to a circular double-stranded DNA into which additional DNA segments can be ligated.
  • a phage vector refers to a viral vector, in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors are episomal mammalian vectors having a bacterial origin of replication).
  • vectors may integrate into the host cell's genome as it enters the host cell, thereby replicating along with the host genome.
  • some vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are termed “recombinant expression vectors” or simply “expression vectors” herein.
  • expression vectors useful in recombinant DNA techniques often exist in the form of plasmids.
  • plasmid and vector may be used interchangeably because the plasmid is the most commonly used form among vectors.
  • the expression vector in the present invention include commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, and Ti vectors; cosmid; phages such as lambda, lambdoid, M13, Mu, p1 P22, Q ⁇ , T-even, T2, T3, and T7; It may be selected from the group consisting of plant viruses, but is not limited thereto, and all expression vectors known to those skilled in the art as expression vectors can be used in the present invention, and when selecting an expression vector, it depends on the properties of the host cell of interest.
  • introducing a vector into a host cell it may be performed by calcium phosphate transfection, viral infection, DEAE-dextran controlled transfection, lipofectamine transfection or electroporation, but is not limited thereto.
  • An introduction method suitable for the expression vector and host cell can be selected and used.
  • the vector contains one or more selection markers, but is not limited thereto, and selection is possible depending on whether a product is produced using a vector that does not contain a selection marker. Selection of the selection marker is selected by the target host cell, and since this method is already known to those skilled in the art, the present invention is not limited thereto.
  • a tag sequence may be inserted into the expression vector and fused thereto.
  • the tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag that facilitates purification known to those skilled in the art can be used in the present invention.
  • a host cell line transformed with the expression vector provided in the present invention is provided.
  • the "host cell” includes an individual cell or cell culture that can be or has been a recipient of vector(s) for incorporation of a polypeptide insert.
  • a host cell includes the progeny of a single host cell, which may not necessarily be completely identical (morphologically or in genomic DNA complement) to the original parent cell because of natural, accidental or deliberate mutations.
  • Host cells include cells transformed in vivo with the polypeptide(s) of the present disclosure.
  • the host cell may include cells of mammalian, plant, insect, fungal or cellular origin, for example, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium; fungal cells such as yeast cells and Pichia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; CHO (Chinese hamster ovary cells), SP2/0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bow melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells) or PERC.6 (Human retinal cells) animal cells; Or it may be a plant cell, but is not limited thereto, and any cell that can be used as a host cell line known to those skilled in the art can be used.
  • bacterial cells such as Escherichia coli,
  • the transformation method of the present invention is any method of injecting a desired vector into the host cell, and may include any known method capable of injecting the vector into the host cell, for example, a method using CaCl 2 , electroporation, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, and transformation using viruses, etc. may be used, but are limited thereto. It is not.
  • a binding molecule specifically binding to the epitope of the present invention is provided.
  • binding molecule of the present invention may be either an antibody or an antigen-binding fragment, but is not limited thereto.
  • the "antibody” is a full-length antibody or a portion of an antibody that has the ability to bind to the Lrig-1 protein and competitively binds to the Lrig-1 antigenic determinant region with the binding molecule of the present invention. All inclusive.
  • the "antibody” refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen.
  • the antigen may be Lrig-1 protein present on the surface of regulatory T cells. Preferably, it may specifically recognize the leucine rich region or immunoglobulin-like domain of the Lrig-1 protein, but is not limited thereto.
  • the "immunoglobulin” has a heavy chain and a light chain, each of which includes a constant region and a variable region.
  • the light chain and heavy chain variable regions include three variable regions called complementarity determining regions (hereinafter referred to as “CDRs”) and four framework regions.
  • CDRs complementarity determining regions
  • the CDR mainly serves to bind to the antigenic determinant (Epitope) of the antigen.
  • the CDRs of each chain are typically referred to sequentially starting from the N-terminus as CDR1, CDR2, and CDR3, and are also identified by the chain in which the particular CDR is located.
  • the antibody or antigen-binding fragment thereof provided herein comprises heavy chain CDRs and light chain CDRs selected from the CDRs provided herein, or includes conservative variants of the CDRs provided herein. It is a chimeric antibody or fragment that
  • variants of an amino acid sequence include amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants.
  • Amino acid deletion variants comprising deletions at the N-terminus and/or C-terminus of a protein are also referred to as N-terminal and/or C-terminal truncation variants.
  • Amino acid insertion variants include insertions of single or two or more amino acids in a particular amino acid sequence.
  • amino acid sequence variants with insertions one or more amino acid residues are inserted at specific sites in the amino acid sequence, but random insertions with appropriate screening of the resulting product are also possible.
  • Amino acid addition variants include amino-terminal and/or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.
  • Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Deletions can be anywhere in the protein.
  • Amino acid substitution variants are characterized in that at least one residue in the sequence is removed and another residue is inserted in its place. It is preferred that the modification is at a position in the amino acid sequence that is not conserved between homologous proteins or peptides and/or replaces an amino acid with another amino acid having similar properties.
  • the amino acid changes in the protein variants are conservative amino acid changes, ie substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitution of one of the family of amino acids in which the side chain is related.
  • Naturally occurring amino acids generally fall into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), and non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan). , and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes grouped together as aromatic amino acids.
  • the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, will be 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% .
  • the degree of similarity or identity is preferably at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% of the full length of the reference amino acid sequence, It is given for an amino acid region that is at least about 80%, at least about 90% or about 100%.
  • the degree of similarity or identity is at least about 20, at least about 40, at least about 60, at least about 80, preferably contiguous amino acids. , at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids.
  • the degree of similarity or identity is given over the full length of a reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably best sequence alignment, for example using standard settings, preferably EMBOSS::needle, Matrix (Matrix). ): Align using Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • Sequence similarity refers to the percentage of amino acids that are identical or exhibit conservative amino acid substitutions.
  • Sequence identity refers to the percentage of identical amino acids between the sequences.
  • percent identity is intended to denote the percentage of identical amino acid residues between two sequences to be compared, obtained after best alignment, this percentage is purely statistical and the differences between the two sequences are randomly distributed and over their full length.
  • Sequence comparison between two amino acid sequences is usually done by optimally aligning these sequences and then comparing them, the comparison being done segment by segment or by "comparison windows” to identify and compare local regions of sequence similarity.
  • Optimal alignment of sequences for comparison may be performed, in addition to manually, by local homology algorithms or by similarity search methods, or by computer programs using these algorithms (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA). Genetics Computer Group's Wisconsin Genetics Software Package (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA) of the Wisconsin Genetics Software Package).
  • Percent identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared, and multiplying the result obtained by 100 to obtain the percentage identity between these two sequences.
  • Homologous amino acid sequences are according to the present invention at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% of the amino acid residues. indicates identity.
  • Amino acid sequence variants described herein can be readily prepared by one skilled in the art, eg, by recombinant DNA manipulation. The manipulation of DNA sequences to produce proteins and peptides with substitutions, additions, insertions or deletions is well known.
  • the peptides and amino acid variants described herein can be readily prepared with the aid of known peptide synthesis techniques, such as, for example, by solid phase synthesis and similar methods.
  • an antibody or antigen-binding fragment thereof provided herein comprises a heavy chain CDR and a light chain CDR selected from the CDRs provided herein, or a humanized antibody comprising conservative variants of the CDRs provided herein, or it's a snippet
  • an antibody or antigen-binding fragment thereof provided herein comprises a light chain and/or heavy chain comprising a sequence provided herein or conservative variants thereof.
  • a conservative variant is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to a reference sequence provided herein. have a sequence
  • conservative variants comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 or more amino acid substitutions, insertions or deletions. do.
  • an antibody that specifically binds to the Lrig-1 protein provided herein comprises a sequence provided herein or a conservative variant thereof.
  • conservative variants include conservative amino acid substitutions, insertions or deletions.
  • conservative amino acid substitutions are substitutions of one amino acid with another amino acid having similar structural or chemical properties, eg, similar side chains, while retaining the biological activity of the reference sequence. Exemplary conservative substitutions are well known in the art.
  • the "full-length antibody” has a structure having two full-length light chains and two full-length heavy chains, each light chain connected to the heavy chain by a disulfide bond, and IgA, IgD, IgE, IgM, and IgG.
  • the IgG includes IgG1, IgG2, IgG3 and IgG4 as its subtype.
  • the "antigen-binding fragment” refers to a fragment having an antigen-binding function
  • examples of antigen-binding fragments include 1 light chain variable region (VL) and heavy chain variable region (VH) and light chain constants.
  • the antigen-binding fragment may be a Fab or F(ab') 2 fragment using a proteolytic enzyme, for example, papain or pepsin, and may be prepared through genetic recombination technology.
  • the antibodies and fragments thereof are monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bivalent ( bivalent), bispecific molecule, minibody, domain antibody, bispecific antibody, antibody mimetic, unibody, diabody, triabody, tetrabody ( tetrabody) or a fragment thereof, but is not limited thereto.
  • the "chimeric antibody” is an antibody in which the variable region of a mouse antibody and the constant region of a human antibody are recombined, and the immune response is greatly improved compared to the mouse antibody.
  • the "humanized antibody” refers to an antibody in which the protein sequence of an antibody derived from a non-human species is modified to resemble an antibody variant naturally produced in a human.
  • the humanized antibody may be prepared by recombination of a mouse-derived CDR with a human antibody-derived FR to prepare a humanized variable region, and then recombination with the constant region of a desired human antibody to prepare a humanized antibody.
  • the binding molecule may be provided as a bispecific antibody or a bispecific antigen-binding fragment capable of binding to Lrig-1 protein and other proteins.
  • the bispecific antibody and bispecific antigen-binding fragment may comprise a binding molecule according to the present invention.
  • the bispecific antibodies and bispecific antigen-binding fragments include an antigen-binding domain capable of binding to Lrig-1 protein, wherein the antigen-binding domain capable of binding to Lrig-1 protein It may comprise or consist of a binding molecule according to the present invention.
  • Bispecific antibodies and bispecific antigen-binding fragments provided by the present invention include an antigen-binding domain, which is a binding molecule capable of binding to the Lrig-1 protein according to the present invention, and an antigen-binding domain capable of binding to other target proteins.
  • the antigen-binding domain capable of binding to another target protein is a protein other than Lrig-1 protein, but is not limited thereto, and may be, for example, an antigen-binding domain capable of binding to PD-1 or a cell surface receptor. However, it is not limited thereto.
  • bispecific antibodies and bispecific antigen-binding fragments according to the present invention may be provided in any suitable format, such as those described in documents incorporated herein by reference in their entirety.
  • a bispecific antibody or bispecific antigen-binding fragment may be a bispecific antibody conjugate (eg an IgG2, F(ab')2 or CovX-body), a bispecific IgG or IgG-like molecule (eg an IgG2, F(ab')2 or CovX-body).
  • Db diabodies
  • dsDb dsDb
  • DART scDb
  • tandAbs tandem scFv
  • tandem dAb/VHH triple body
  • Fab-scFv Fab-scFv
  • F(ab')2-scFv2 bispecific Fc and CH3 fusion proteins
  • taFv-Fc di-diabody, scDb-CH3, scFv-Fc-scFv, HCAb-VHH, scFv-kih-Fc, or scFv-kih-CH3), or a bispecific fusion protein such as scFv2-albumin , scDb-albumin, taFv-toxin, DNL-Fab3, DNL-Fab4-IgG, DNL-Fab4-IgG-cytokine 2).
  • scFv2-albumin scDb-albumin
  • taFv-toxin DNL-Fab3, DNL-Fab4-IgG, DNL-Fab4-IgG-cytokine 2
  • the method for producing the bispecific antibody in the present invention includes chemical cross-linking of an antibody or antibody fragment with a reducing disulfide or non-reducing thioether bond.
  • a reducing disulfide or non-reducing thioether bond For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) is used to generate disulfide-linked bispecific F(ab)2 heterodimers, for example, It can be used to chemically cross-link Fab fragments via hinge region SH-groups.
  • SPDP N-succinimidyl-3-(-2-pyridyldithio)-propionate
  • another method for producing the bispecific antibody in the present invention is to fuse an antibody-producing hybridoma with, for example, polyethylene glycol to create a quadroma cell capable of secreting the bispecific antibody.
  • Bispecific antibodies and bispecific antigen-binding fragments according to the present invention can be produced recombinantly, for example by expression from a nucleic acid construct encoding a polypeptide for the antigen-binding molecule.
  • two antigen binding domains i.e., light and heavy chain variable domains for an antigen binding domain capable of binding PD-1, etc., and light and heavy chain variable domains for an antigen binding domain capable of binding other target proteins.
  • a DNA construct comprising sequences encoding the light and heavy chain variable domains for ) and a suitable linker between the antigen binding domains or a dimerization domain can be prepared by molecular cloning techniques.
  • Recombinant bispecific antibodies may then be produced by expression (eg in vitro) of the construct in a suitable host cell (eg mammalian host cell), and the expressed recombinant bispecific antibody may then optionally be purified.
  • An antibody may be produced by an affinity maturation process that results in a modified antibody with improved affinity of the antibody for an antigen compared to the unmodified parental antibody.
  • Affinity matured antibodies can be produced by procedures known in the art.
  • the binding molecule provided in the present invention may include a variant of the amino acid sequence as long as it can specifically bind to the Lrig-1 protein.
  • changes may be made to the amino acid sequence of an antibody to improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of residues in the amino acid sequence of the antibody.
  • amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc.
  • Analysis of the size, shape and type of amino acid side chain substituents revealed that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine can be said to be biologically functional equivalents.
  • each amino acid is given a hydrophobicity index according to its hydrophobicity and charge: Isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); Tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); Lysine (-3.9); and arginine (-4.5).
  • the hydrophobic amino acid index is very important in conferring the interactive biological function of proteins. It is a known fact that amino acids having similar hydrophobicity indexes should be substituted to retain similar biological activities. When a mutation is introduced with reference to the hydrophobicity index, substitution is made between amino acids exhibiting a difference in hydrophobicity index, preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • Amino acid exchanges in proteins that do not entirely alter the activity of the molecule are known in the art.
  • the most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, and Gln/Glu.
  • the binding molecule of the present invention is construed to include sequences showing substantial identity with the sequences listed in the Sequence Listing.
  • the term "substantial identity” refers to a sequence of at least 61% when the sequence of the present invention and any other sequence are paralleled so as to correspond as much as possible, and the paralleled sequence is analyzed using an algorithm commonly used in the art. It means a sequence exhibiting homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology.
  • Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment can be accessed from NCBI Basic Local Alignment Search Tool (BLAST), NBCI (National Center for Biological Information), etc. available. BLAST is accessible at this address (www.ncbi.nlm.nih.gov/BLAST/). A sequence homology comparison method using this program can be checked online (www.ncbi.nlm.nih.gov/BLAST/blast_help.html).
  • the binding molecule preferably the antibody
  • the binding molecule may be produced by a conventional method for producing antibodies, but may also be produced by affinity maturation.
  • the affinity maturation refers to a process in which activated B cells produce antibodies with increased affinity for an antigen during an immune response.
  • the affinity maturation may generate antibodies or antibody fragments produced by affinity maturation based on the principle of mutation and selection, just like a process that occurs in nature.
  • the "monoclonal antibody” refers to an antibody molecule of a single molecular composition obtained from substantially the same antibody population, and exhibits a single binding specificity and affinity for a specific epitope.
  • the "binding" or “specific binding” refers to the affinity of the antibody or antibody composition for the antigen of the present application.
  • “Specific binding” in antigen-antibody binding can typically be distinguished from non-specific background binding if the dissociation constant (Kd) is less than 1x10 -5 M or less than 1x10 -6 M or less than 1x10 -7 M.
  • Specific binding can be detected by methods known in the art, such as ELISA, surface plasmon resonance (SPR), immunoprecipitation, coprecipitation, and the like, and non-specific binding and specific binding Include an appropriate control group that can be differentiated.
  • the antibody or antigen-binding fragment of the present invention may exist as a multimer, such as a dimer, a trimer, a tetramer, or a pentamer, including at least a portion of the antigen-binding ability of a monomer.
  • These multimers also include homomultimers or heteromultimers. Since antibody multimers contain multiple antigen-binding sites, they have superior antigen-binding ability compared to monomers. Multimers of antibodies are also easy to construct multifunctional (bifunctional, trifunctional, tetrafunctional) antibodies.
  • multifunctional refers to an antibody or antigen-binding fragment having two or more activities or functions (eg, antigen-binding ability, enzyme activity, ligand or receptor binding ability).
  • the antibody of the present invention Polypeptides having enzymatic activity, for example, luciferase, acetyltransferase, galactosidase and the like can be coupled.
  • Multifunctional antibodies also include antibodies in the form of multivalent or multispecific (bispecific, trispecific, etc.).
  • the antibody or antigen-binding fragment according to the present invention specifically binds to an epitope comprising a polypeptide represented by any one of the amino acid sequences of SEQ ID NOs: 35 to 45 present on regulatory T cells, thereby inhibiting the function of the regulatory T cells.
  • various diseases such as immune-related diseases; Alternatively, neurodegenerative diseases or neuroinflammatory diseases can be prevented or treated.
  • the “immune-related disease” of the present invention may be at least one selected from the group consisting of autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute or chronic inflammatory disease, but is limited thereto It is not.
  • the autoimmune disease of the present invention is rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behçetsi disease, Sjögren's syndrome, Guilla-Barré syndrome, chronic thyroiditis, multiple sclerosis , polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa, but may be at least one selected from the group consisting of, but is not limited thereto.
  • “Cranial nervous system disease” for preventing, improving or treating the composition provided in the present invention may be a neurodegenerative disease or a neuroinflammatory disease.
  • neurodegenerative disease and “neuroinflammatory disease” of the present invention include stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease , prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, AlzAmyotrophic lateral sclerosis, Paraneoplastic syndrome, corticobasal degeneration, Select from the group consisting of systemic atrophy, progressive supranuclear palsy, autoimmune disease of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury and tauopathy It may be, but is not limited thereto.
  • a binding molecule, antibody or antigen-binding fragment provided by the present invention and an antibody-drug conjugate (Antibody-Drug Conjugate, ADC) comprising a drug is provided.
  • ADC Antibody-Drug Conjugate
  • the "antibody-drug conjugate (ADC)" refers to a form in which a drug and an antibody are chemically linked without reducing the biological activity of the antibody and the drug.
  • the antibody-drug conjugate is a form in which a drug is bound to an amino acid residue at the N-terminus of the heavy and/or light chain of an antibody, specifically, a drug to an ⁇ -amine group at the N-terminus of the heavy and/or light chain of an antibody. refers to this combined form.
  • the "drug” may mean any substance having a specific biological activity in cells, which is a concept including DNA, RNA, or peptide.
  • the drug may be in a form containing a reactive group capable of reacting with an ⁇ -amine group and cross-linking, and also includes a form in which a linker including a reactive group capable of reacting with an ⁇ -amine group and cross-linking is connected.
  • the type is not particularly limited. It includes all types that react with known amine groups. Examples include Isothiocyanate, Isocyanates, Acyl Azide, NHS ester, Sulfonyl chloride, Aldehyde, Glyoxal, Epoxide, Oxirane, Carbonate, Aryl halide, Imidoester, Carbodiimide, Anhydride and Fluorophenyl ester), but is not limited thereto.
  • the antibody-drug conjugate is the antibody or antigen-binding fragment comprising the epitope of the present invention, that is, Lrig-1 protein; or an epitope comprising a polypeptide consisting of a partial amino acid sequence of an extracellular domain of Lrig-1 protein; or an antibody or antigen-binding fragment that specifically binds to an epitope comprising a polypeptide represented by any one of amino acid sequences of SEQ ID NOs: 7 to 17, wherein the drug is an antibody that specifically binds to Lrig-1 protein
  • Any drug capable of treating a disease targeted by may be included, for example, a drug capable of treating an immune-related disease; Alternatively, it may be a drug capable of treating neurodegenerative diseases or neuroinflammatory diseases, but is not limited thereto.
  • the antibody or antigen-binding fragment provided by the present invention or antibody-drug conjugates (Antibody-Drug Conjugate, ADC) as an active ingredient, including various diseases, for example, immune-related diseases; Or it provides a pharmaceutical composition for the prevention or treatment of brain nervous system disease.
  • ADC Antibody-Drug Conjugate
  • a binding molecule, antibody or antigen-binding fragment included as an active ingredient in the pharmaceutical composition in the present invention specifically binds to an epitope comprising a polypeptide represented by any one of the amino acid sequences of SEQ ID NOs: 35 to 45 to regulate the function of the regulatory T cells, and to effector T cells Through the process of regulating activity, various diseases can be treated very effectively.
  • the “immune-related disease” of the present invention may be at least one selected from the group consisting of autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute or chronic inflammatory disease, but is limited thereto It is not.
  • the autoimmune disease of the present invention is rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behçetsi disease, Sjögren's syndrome, Guilla-Barré syndrome, chronic thyroiditis, multiple sclerosis , polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa, but may be at least one selected from the group consisting of, but is not limited thereto.
  • “Cranial nervous system disease” for preventing, improving or treating the composition provided in the present invention may be a neurodegenerative disease or a neuroinflammatory disease.
  • neurodegenerative disease and “neuroinflammatory disease” of the present invention include stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease , prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, AlzAmyotrophic lateral sclerosis, Paraneoplastic syndrome, corticobasal degeneration, Select from the group consisting of systemic atrophy, progressive supranuclear palsy, autoimmune disease of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury and tauopathy It may be, but is not limited thereto.
  • an immune-related disease comprising, as an active ingredient, a Chimeric Antigen Receptor (CAR) comprising an antigen-specific binding domain, a linking domain, and a CD3 zeta ( ⁇ ) signaling domain, or Provided is a pharmaceutical composition for preventing or treating brain nervous system diseases.
  • CAR Chimeric Antigen Receptor
  • ⁇ CD3 zeta
  • chimeric antigen receptor means an engineered receptor comprising an extracellular antigen binding domain and an intracellular signaling domain.
  • CAR single chain variable fragment
  • the binding domain may include a single chain variable fragment (scFv) capable of specifically recognizing the Lrig-1 protein.
  • scFv single chain variable fragment
  • the "single chain variable fragment” or “scFv” refers to a fusion protein of a variable heavy chain (VH) and a variable light chain (VL) of an antibody by a peptide linker between VL and VH.
  • the VH domain and the VL domain may be connected through a flexible linker.
  • the flexible linker is about 10 to 30 amino acids (eg, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids), preferably 15 amino acids long.
  • the linker length can act as an important determining site for chimeric antigen receptors, so a linker shorter than the above range can increase affinity, but also cause intracellular multimer formation to impair CAR expression. On the other hand, linkers longer than this range may reduce antigen affinity by moving the VL and VH CDRs farther apart in space.
  • the chimeric antigen receptor of the present invention may further include at least one of a hinge region (or spacer) and a signaling domain.
  • the hinge region is a part connecting the antigen-binding domain and the transmembrane domain, and is also called a 'spacer', and has the purpose of extending the antigen-binding domain from the T cell membrane or the NK cell membrane.
  • the hinge region can be obtained from any suitable sequence from any genus, including, for example, human or parts thereof, or CD8, CD28, 4-1BB, OX40 commonly used in the art.
  • the hinge region may include one selected from immunoglobulins (eg, IgG1, IgG2, IgG3, IgG4, and IgD) without being limited to immunoglobulins, but is not limited thereto.
  • immunoglobulins eg, IgG1, IgG2, IgG3, IgG4, and IgD
  • the signaling domain refers to a part of a chimeric antigen receptor found or engineered to be found inside a T cell.
  • the signaling domain may or may not include a transmembrane domain that serves to fix the chimeric antigen receptor in the plasma membrane of T cells.
  • the transmembrane domain and the signaling domain may be derived from the same protein (eg, CD3 zeta ( ⁇ ) molecule), or the transmembrane domain and the signaling domain may be derived from different proteins (eg, CD3 zeta ( ⁇ ) molecule).
  • CD3 zeta ( ⁇ ) molecule the transmembrane domain of CD28 and the intracellular signaling domain of the CD3 zeta ( ⁇ ) molecule, or vice versa).
  • the transmembrane domain includes, for example, T cell receptor ⁇ or ⁇ chain, all or part of the CD3 zeta ( ⁇ ) chain, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof, but is not limited thereto.
  • the costimulatory domain comprises 4-1BB (CD137); OX40; CD27; CD28; CD30; CD40; PD-1; CD2; CD7; CD258; natural killer group 2 member C (NKG2C); natural killer group 2 member (NKG2D); B7-H3; CD83; ICAM-1; ligands that bind to LFA-1 (CD11a/CD18) or ICOS; active fragments thereof; functional derivatives thereof; or a functional signaling domain derived from a polypeptide comprising a combination thereof, but is not limited thereto.
  • 4-1BB CD137
  • OX40 CD27; CD28; CD30; CD40; PD-1; CD2; CD7; CD258; natural killer group 2 member C (NKG2C); natural killer group 2 member (NKG2D); B7-H3; CD83; ICAM-1; ligands that bind to LFA-1 (CD11a/CD18) or ICOS; active fragments thereof; functional derivatives thereof; or a
  • the signaling domain is all or part of CD3 zeta ( ⁇ ), common FcR gamma (FcER1G), FcgammaRIIIa, FcRbeta (Fc epsilon lip), CD3gamma, CD3delta, CD3 epsilon, CD79a, CD79b,
  • DAP10 DNAX-activating protein 10
  • DAP12 DNAX-activating protein 12
  • an active fragment thereof a functional derivative thereof, or a combination thereof
  • the “immune-related disease” of the present invention may be at least one selected from the group consisting of autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute or chronic inflammatory disease, but is limited thereto It is not.
  • the autoimmune disease of the present invention is rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behçetsi disease, Sjögren's syndrome, Guilla-Barré syndrome, chronic thyroiditis, multiple sclerosis , polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa, but may be at least one selected from the group consisting of, but is not limited thereto.
  • “Cranial nervous system disease” for preventing, improving or treating the composition provided in the present invention may be a neurodegenerative disease or a neuroinflammatory disease.
  • neurodegenerative disease and “neuroinflammatory disease” of the present invention include stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease , prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, AlzAmyotrophic lateral sclerosis, Paraneoplastic syndrome, corticobasal degeneration, Select from the group consisting of systemic atrophy, progressive supranuclear palsy, autoimmune disease of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury and tauopathy It may be, but is not limited thereto.
  • prevention may include without limitation any act of blocking or suppressing or delaying the symptoms of a disease by using the pharmaceutical composition of the present invention.
  • treatment may include without limitation any action that improves or benefits the symptoms of a disease by using the pharmaceutical composition of the present invention.
  • the pharmaceutical composition may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be intended for humans.
  • the pharmaceutical compositions are not limited thereto, but are formulated in the form of oral formulations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively.
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. for oral administration, and buffers, preservatives, and painless agents for injections.
  • the dosage form of the pharmaceutical composition of the present invention may be variously prepared by mixing with a pharmaceutically acceptable carrier as described above.
  • a pharmaceutically acceptable carrier for example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. there is.
  • it may be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, and the like.
  • examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, and the like can be used.
  • fillers, anti-agglomerating agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like may be further included.
  • the route of administration of the pharmaceutical composition in the present invention is not limited to these, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, This includes sublingual or rectal. Oral or parenteral administration is preferred.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.
  • the pharmaceutical composition of the present invention depends on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, route of administration, excretion rate, drug combination and severity of the specific disease to be prevented or treated. It can vary widely, and the dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, degree of disease, drug type, administration route and period, but can be appropriately selected by those skilled in the art, and is 0.0001 to 50 mg/kg per day. Alternatively, it may be administered at 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way.
  • the pharmaceutical composition according to the present invention may be formulated into a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.
  • a binding molecule according to the present invention or antibody-drug conjugates (Antibody-Drug Conjugate, ADC); or administering a pharmaceutically effective amount of a Chimeric Antigen Receptor (CAR) to a subject, for example, immune-related diseases; Or it relates to a method for preventing or treating a brain nervous system disease.
  • ADC Antibody-Drug Conjugate
  • CAR Chimeric Antigen Receptor
  • the “immune-related disease” of the present invention may be at least one selected from the group consisting of autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute or chronic inflammatory disease, but is limited thereto It is not.
  • the autoimmune disease of the present invention is rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behçetsi disease, Sjögren's syndrome, Guilla-Barré syndrome, chronic thyroiditis, multiple sclerosis , polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa, but may be at least one selected from the group consisting of, but is not limited thereto.
  • “Cranial nervous system disease” for preventing, improving or treating the composition provided in the present invention may be a neurodegenerative disease or a neuroinflammatory disease.
  • neurodegenerative disease and “neuroinflammatory disease” of the present invention include stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease , prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, AlzAmyotrophic lateral sclerosis, Paraneoplastic syndrome, corticobasal degeneration, Select from the group consisting of systemic atrophy, progressive supranuclear palsy, autoimmune disease of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury and tauopathy It may be, but is not limited thereto.
  • the antibody-drug conjugate specifically binds to an epitope comprising a polypeptide represented by any one of the amino acid sequences of SEQ ID NOs: 35 to 45 to regulate the function of the regulatory T cell and regulate the activity of the effector T cell, thereby producing various The disease can be treated very effectively.
  • the "individual” is an individual suspected of having an immune-related disease or a brain nervous system disease
  • the subject suspected of having an immune-related disease or a brain nervous system disease is a mouse, including a human who has or may develop the disease, It refers to mammals including livestock, etc., but subjects that can be treated with the antibody, antigen-binding fragment or antibody-drug conjugate of the present invention are included without limitation.
  • the method of the present invention may include administering a pharmaceutically effective amount of an antibody or antibody-drug conjugate.
  • An appropriate total daily amount can be determined by a treating physician within the scope of sound medical judgment, and can be administered once or divided into several times.
  • a specific therapeutically effective amount for a particular patient is determined by the type and extent of the response to be achieved, the specific composition, including whether other agents are used as the case may be, the patient's age, weight, general state of health, It is preferable to apply differently according to various factors including gender and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together with or concurrently used with a specific composition, and similar factors well known in the medical field.
  • the method for preventing or treating the disease may be a combination therapy further comprising administering a compound or substance having therapeutic activity for one or more diseases.
  • the "combination" should be understood to denote simultaneous, separate or sequential administration.
  • the interval between administrations of the second components should be such that the beneficial effects of the combination are not lost.
  • the administration dose of the antibody or antibody-drug conjugate may be about 0.0001 ⁇ g to 500 mg per 1 kg of patient body weight, but is not limited thereto.
  • Covalent Labeling MS CL-MS
  • XL-MS Cross-linking MS
  • the protein used in the screening method of the present invention may be a form displayed on the cell surface, a form displayed on the surface of a virus (eg, bacteriophage), an isolated form, or a purified form. there is.
  • a protein displayed on the surface of a cell or virus it is preferable to immobilize the cell or virus on a solid substrate for expedited or automated screening.
  • immobilize the protein in an isolated or purified form on a solid substrate Any material commonly used in the art may be used as the substrate, and examples thereof include, but are not limited to, hydrocarbon polymers such as polystyrene and polypropylene, glass, metal, and gel.
  • Solid phase substrates can be provided in the form of dipsticks, microtiter plates, particles (eg beads), affinity columns and immunoblot membranes (eg polyvinylidene fluoride membranes). Most preferably, the solid substrate is a microtiter plate.
  • the screening method of the present invention can be carried out in various ways, and in particular, it can be carried out in a high throughput manner according to various binding assays known in the art.
  • the test substance or the proteins may be labeled with a detectable label.
  • the detectable label may be a chemical label (e.g., biotin), an enzyme label (e.g., horseradish peroxidase, alkaline phosphatase, peroxidase, luciferase, ⁇ -galacto) sidase and ⁇ -glucosidase), radioactive labels (e.g., C14, I125, P32 and S35), fluorescent labels (e.g., coumarin, fluorescein, fluoresein isothiocyanate (FITC), rhodamine 6G, rhoda Min B (rhodamine B), TAMRA (6-carboxy-tetramethyl-rhodamine), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino-2phenylindole), HEX, TET, Dabsyl and FAM
  • a chemical label e.g
  • the occurrence of binding between the protein and the test substance can be analyzed by detecting a signal from the label.
  • alkaline phosphatase bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS-B1-phosphate ) and chromogenic substrates such as ECF (enhanced chemifluorescence) to detect signals.
  • BCIP bromochloroindolyl phosphate
  • NBT nitro blue tetrazolium
  • naphthol-AS-B1-phosphate naphthol-AS-B1-phosphate
  • chromogenic substrates such as ECF (enhanced chemifluorescence)
  • an epitope of Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein at least one selected from the group consisting of polypeptides consisting of amino acid sequences represented by SEQ ID NO: 35 to SEQ ID NO: 45 Provides an epitope comprising a polypeptide of
  • polypeptide provides an epitope, consisting of the amino acid sequence represented by SEQ ID NO: 38 or 44.
  • the polypeptide provides an epitope consisting of the amino acid sequences represented by SEQ ID NOs: 35 to 37, 39 to 43, and 45.
  • Another embodiment of the present invention provides a nucleic acid molecule encoding the epitope.
  • Another embodiment of the present invention provides an expression vector into which the nucleic acid molecule is inserted.
  • the expression vector provides a host cell line transfected
  • One embodiment of the present invention provides a binding molecule that specifically binds to an epitope containing at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences represented by SEQ ID NO: 35 to SEQ ID NO: 45.
  • Another embodiment of the present invention provides a binding molecule, wherein the binding molecule is an antibody or a fragment thereof.
  • the antibody is a chimeric antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, or an antibody mimetic. , a diabody, a triabody, a tetrabody, or a fragment thereof, to provide a binding molecule.
  • the antigen-specific binding domains are SEQ ID NOs: 35 to 45
  • An antigen-specific binding domain that specifically binds to an epitope comprising at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequence shown; and a chimeric antigen receptor which is a fusion protein comprising an immunoglobulin Fc region.
  • Another embodiment of the present invention provides an antibody-drug conjugate comprising the above binding molecule and drug.
  • Another embodiment of the present invention provides a pharmaceutical composition for preventing or treating immune-related diseases comprising any one of the binding molecule, chimeric antigen receptor, and antibody-drug conjugate as an active ingredient.
  • the immune-related disease is at least one selected from the group consisting of autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute or chronic inflammatory disease. It provides a pharmaceutical composition for preventing or treating related diseases.
  • a pharmaceutical composition for preventing or treating brain nervous system diseases comprising any one of the binding molecule, the chimeric antigen receptor, and the antibody-drug conjugate as an active ingredient.
  • the brain nervous system disease is a neurodegenerative disease or a neuroinflammatory disease, provides a pharmaceutical composition.
  • the neurodegenerative disease or neuroinflammatory disease is stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease , prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, AlzAmyotrophic lateral sclerosis, Paraneoplastic syndrome, corticobasal degeneration, Selected from the group consisting of systemic atrophy, progressive supranuclear palsy, autoimmune disease of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury and tauopathy It provides a pharmaceutical composition for the prevention or treatment of at least one, neurodegenerative disease or neuroinflammatory disease.
  • Another embodiment of the present invention provides a method for screening a protein epitope of Lrig-1 (leucine-rich and immunoglobulin-like domains 1) comprising the following steps.
  • (a) preparing an antibody comprising the heavy chain region represented by the amino acid sequence of SEQ ID NO: 17 and the light chain region represented by the amino acid sequence of SEQ ID NO: 18 or the heavy chain region represented by the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20 Preparing an antibody comprising a light chain region represented by the amino acid sequence of or preparing an antibody comprising a heavy chain region represented by the amino acid sequence of SEQ ID NO: 21 and a light chain region represented by the amino acid sequence of SEQ ID NO: 22
  • Another embodiment of the present invention provides a method for screening an epitope of Lrig-1 protein using the antibody.
  • Another embodiment of the present invention provides a method for screening a binding molecule that is an antibody or a fragment thereof using the epitope.
  • Another embodiment of the present invention provides a method for binding the epitope with a binding molecule that is an antibody or a fragment thereof.
  • a binding molecule that is an antibody or a fragment thereof that specifically binds to Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein,
  • the heavy chain variable region (VH) of the binding molecule comprises: the amino acid sequence represented by SEQ ID NOs: 46, 52, 58, 64, 70, 76, 82 and 88; Heavy chain variable region (VH) CDR1 selected from the group consisting of; a heavy chain variable region (VH) CDR2 selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 47, 53, 59, 65, 71, 77, 83 and 89; and a heavy chain variable region (VH) CDR3 selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 48, 54, 60, 66, 72, 78, 84 and 90; and
  • VL light chain variable region
  • VL light chain variable region
  • Heavy chain variable region (VL) CDR1 selected from the group consisting of; a heavy chain variable region (VL) CDR2 selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 50, 56, 62, 68, 74, 80, 86 and 92; and a heavy chain variable region (VL) CDR3 selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 51, 57, 63, 69, 75, 81, 87 and 93; ego
  • the binding molecule provides a binding molecule that specifically binds to an epitope containing at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences represented by SEQ ID NO: 35 to SEQ ID NO: 45.
  • the antibody or antigen-binding fragment specifically binding to the epitope of Lrig-1 protein according to the present invention inhibits the function of regulatory T cells by specifically binding to the epitope of the present invention present on regulatory T cells, and By maintaining or increasing the activity of T cells, it can be used very efficiently for the prevention, improvement or treatment of various diseases.
  • FIG 1 shows the structure of Lrig-1 protein according to an embodiment of the present invention.
  • FIG. 2 shows the structure of Lrig-1 protein according to an embodiment of the present invention.
  • Figure 3 shows the expression level of Lrig-1 mRNA according to an embodiment of the present invention.
  • Figure 4 shows the expression level of Lrig-1 mRNA according to an embodiment of the present invention.
  • FIG. 5 shows the expression level of Lrig-1 mRNA according to an embodiment of the present invention.
  • FIG. 6 shows the expression levels of Lrig-1, Lrig-2 and Lrig-3 mRNA according to an embodiment of the present invention.
  • FIG. 7 shows results of comparing the expression level of Lrig-1 protein in regulatory T cells and non-regulatory T cells according to an embodiment of the present invention.
  • FIG 8 shows the expression of Lrig-1 protein on the surface of regulatory T cells according to an embodiment of the present invention.
  • FIG. 9 is a diagram showing antibodies (GTC210-01, GTC210-02, GTC210-03, GTC210-04, GTC110-01, GTC110-02, GTC110-03 and GTC110-04) and Lrig-1 protein in one embodiment of the present invention. It shows the result of analyzing the binding force for .
  • FIG. 10 shows monoclonal antibodies (GTC210-01, GTC210-02, GTC210-03, GTC210-04, GTC110-01, GTC110-02, GTC110-03 and GTC110) specific for the Lrig-1 protein in an embodiment of the present invention. -04) and the regulatory mechanism of Lrig-1 protein-induced Stat3 phosphorylation in regulatory T cells.
  • FIG. 11 shows the result of confirming the weight loss after intraperitoneal injection of the monoclonal antibody according to the present invention to the inflammatory bowel disease animal model according to an embodiment of the present invention.
  • FIG. 12 shows an experimental design diagram for producing a multiple sclerosis animal model according to an embodiment of the present invention.
  • FIG. 13 is a graph showing the treatment effect evaluation score after injecting the monoclonal antibody according to the present invention into an animal model of multiple sclerosis according to an embodiment of the present invention.
  • FIG. 14 is a graph showing the treatment effect evaluation score after injecting the monoclonal antibody according to the present invention into an animal model for multiple sclerosis according to an embodiment of the present invention.
  • Figure 15 shows that it was confirmed that demyelination was remarkably reduced when the antibody according to an embodiment of the present invention was administered.
  • helper T cell 1 CD4+ T-bet+
  • helper T cell 17 CD4+ RoR ⁇ t+
  • regulatory T cell CD4+ Foxp3+
  • various inflammatory cytokines IFN-gamma, IL-17A
  • anti-inflammatory cytokine IL-10 increased expression.
  • FIG. 17 to 20 are Y-maze test (FIG. 17), novel object, treatment effect by injection of monoclonal antibody according to the present invention in each group (G1 to G4) of Alzheimer's animal model according to an embodiment of the present invention.
  • FIG. 21 to 23 show peptides degraded by treatment with proteases (chymotrypsin (FIG. 21), trypsin (FIG. 22) and Asp-N/Lys-C (FIG. 23)) according to an embodiment of the present invention. It shows the results confirmed through the labeling analysis.
  • proteases chymotrypsin (FIG. 21), trypsin (FIG. 22) and Asp-N/Lys-C (FIG. 23)
  • 24 and 25 show the results of aligning and comparing peptides degraded by each protease according to an embodiment of the present invention based on sequences identified by covalant labeling analysis.
  • FIG. 26 and 27 show the results of cross-linking analysis of peptides digested by treatment with proteases (chymotrypsin (FIG. 26) and trypsin (FIG. 27)) according to an embodiment of the present invention.
  • 28 and 29 show the results of aligning and comparing peptides degraded by each protease according to an embodiment of the present invention based on sequences identified by cross-linking analysis.
  • FIG. 30 shows the results of aligning and analyzing epitopes identified by covalant labeling and cross-linking analysis according to an embodiment of the present invention.
  • the present invention relates to an epitope of Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein, which is an antigen present on the surface of regulatory T cells, and an antibody or antigen-binding fragment specifically binding thereto.
  • Lrig-1 leucine-rich and immunoglobulin-like domains 1
  • an epitope of the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein comprising the amino acid sequence represented by SEQ ID NOs: 38, 39, 41, 42, 44 and 45
  • An epitope selected from the group consisting of polypeptides is provided.
  • a binding molecule that specifically binds to an epitope containing at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences represented by SEQ ID NO: 35 to SEQ ID NO: 45 is provided. .
  • the antigen-specific binding domain is SEQ ID NO: 35 to SEQ ID NO: 35
  • An antigen-specific binding domain that specifically binds to an epitope comprising at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequence represented by 45; and a chimeric antigen receptor which is a fusion protein comprising an immunoglobulin Fc region.
  • binding molecule that is an antibody or a fragment thereof that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein
  • the heavy chain variable region (VH) of the binding molecule comprises: the amino acid sequence represented by SEQ ID NOs: 46, 52, 58, 64, 70, 76, 82 and 88; Heavy chain variable region (VH) CDR1 selected from the group consisting of; a heavy chain variable region (VH) CDR2 selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 47, 53, 59, 65, 71, 77, 83 and 89; and a heavy chain variable region (VH) CDR3 selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 48, 54, 60, 66, 72, 78, 84 and 90; and
  • VL light chain variable region
  • VL light chain variable region
  • Heavy chain variable region (VL) CDR1 selected from the group consisting of; a heavy chain variable region (VL) CDR2 selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 50, 56, 62, 68, 74, 80, 86 and 92; and a heavy chain variable region (VL) CDR3 selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 51, 57, 63, 69, 75, 81, 87 and 93; ego
  • the binding molecule provides a binding molecule that specifically binds to an epitope containing at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences represented by SEQ ID NO: 35 to SEQ ID NO: 45.
  • T cell subsets such as Th0, Th1, Th2, Th17 and iTreg were prepared.
  • the iTreg refers to cells artificially induced to differentiate in a medium containing the following composition.
  • T cells As for the subtype of T cells, first, naive T cells obtained from the spleen of mice were isolated, and RPMI1640 (Invitrogen Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS; hyclone, logan, UT) was used. Differentiation was induced into each cell through 72-hour culture in a 37°C, 5% CO 2 incubator by further including the components of Table 1 below in the nutrient medium, respectively.
  • FBS fetal bovine serum
  • LRR1 to LRR15 were present in the Lrig-LRR domain (amino acid sequences 41 to 494) among the extracellular domains of the Lrig-1 protein.
  • LRR domains consisted of 23 to 27 amino acids, and 3 to 5 leucines were present.
  • Lrig-1 protein could act as a biomarker specific to regulatory T cells.
  • CD4 + T cells were isolated from the spleen of mice using CD4 beads using magnet-activated cell sorting (MACS). Thereafter, regulatory T (CD4 + CD25 + T) cells and non-regulatory T (CD4 + CD25 - T) cells were isolated using a fluorescence-activated cell sorter (FACS) using the CD25 antibody.
  • FACS fluorescence-activated cell sorter
  • mRNA was extracted from each cell and the cells differentiated in Preparation Example 1 using Trizol, and gDNA was removed from genomic RNA using a gDNA extraction kit (Qiagen) according to the protocol provided by the company. .
  • mRNA from which gDNA was removed was synthesized into cDNA using the BDsprint cDNA synthesis kit (Clonetech).
  • the real-time polymerase chain reaction was carried out using SYBR Green (Molecular Probes) under the conditions of 40 cycles of 3 minutes at 95 ° C, 15 seconds at 61 ° C, and 30 seconds at 72 ° C according to the protocol provided by the company. It was performed using primers, and the relative gene expression level was calculated using the ⁇ CT method, and normalized using HPRT, and the results are shown in FIGS. 3 to 6.
  • Lrig-1 is 18.1 times higher in regulatory T (CD4 + CD25 + T) cells than in non-regulatory T (CD4 + CD25 - T) cells. This level was about 10 times higher than that of Lag3 and Ikzf4, which are known markers of regulatory T cells.
  • regulatory T cells are higher than those of other types of immune cells. Expression of Lrig-1 mRNA was significantly higher in cells, especially in naturally isolated regulatory T cells (nTreg) compared to induced regulatory T cells (iTreg).
  • Lrig-1 was the highest among Lrig-1, Lrig-2, and Lrig-3 corresponding to the Lrig family.
  • the Lrig-1 protein according to the present invention is specifically expressed in regulatory T cells, particularly in naturally occurring regulatory T cells.
  • Lrig-1 protein expressed from Lrig-1 mRNA was specifically expressed only in regulatory T cells.
  • non-regulatory T cells indicated by dotted lines showed almost the same level of Lrig-1 as that of the negative control group, but many regulatory T cells showed high levels of Lrig-1.
  • the Lrig-1 protein according to the present invention is specifically expressed in regulatory T cells.
  • Lrig-1 protein In order for Lrig-1 protein to be a target for cell therapy, it must be expressed on the surface of regulatory T cells for more effective targeted therapy. Therefore, whether Lrig-1 protein was expressed on the surface was checked.
  • Each differentiated T cell subtype of Preparation Example 1 was stained with anti-CD4-APC and anti-Lrig-1-PE antibodies, and each cell surface was stained using a fluorescence-activated cell sorter (FACS). The expression level of Lrig-1 was measured in , and the results are shown in FIG. 8 .
  • FACS fluorescence-activated cell sorter
  • Lrig-1 was expressed in an amount of 0.77 to 15.3 in activated T cells, Th1 cells, Th2 cells, Th17 cells, and Naive T cells, whereas differentiation was expressed as high as 83.9 in induced T cells (iTreg).
  • the Lrig-1 protein according to the present invention is not only specifically expressed in regulatory T cells (Treg) cells, but also has a higher level of expression, particularly on the surface of regulatory T cells.
  • An antibody specific to the Lrig-1 protein according to the present invention was prepared. This antibody was not produced by specifying a specific antigenic determinant, but an antibody capable of binding to any site on the Lrig-1 protein was produced.
  • the antibody cells expressing the Lrig-1 protein were prepared. More specifically, the DNA fragment corresponding to SEQ ID NO: 2 and pcDNA (hygro) are cut with a cutting enzyme, incubated at 37 ° C., and then ligated to insert the DNA sequence of the Lrig-1 protein The prepared pcDNA was prepared. The prepared pcDNA into which SEQ ID NO: 2 was inserted was introduced into L cells through transfection so that Lrig-1 protein could be expressed on the surface of L cells.
  • Light chain and heavy chain amino acid sequences capable of binding to Lrig-1 expressed on the cell surface were selected from the Human scFv library, and a total of 8 heavy and light chains were selected.
  • a monoclonal antibody was prepared by fusing the selected heavy and light chain amino acid sequences with the mlgG2a Fc region or the human IgG1 Fc region.
  • the sequences of the monoclonal antibodies are shown in Table 3 below.
  • Preparation Examples 1 to 9 recognize Lrig-1 well, the antibodies of Preparation Examples 1 to 9 are bound to L cells stably expressing Lrig-1. After that, a secondary antibody capable of recognizing the mouse antibody and conjugated to eFlour 670 was added, and then the binding ability of the above preparations to the Lrig-1 protein was analyzed using FACS to help the result. 9.
  • Preparation Examples 1 to 9 affect the signal transduction pathway in regulatory T cells through the Lrig-1 protein
  • the monoclonal antibodies corresponding to Preparation Examples 1 to 9 After treating regulatory T cells with antibodies to stimulate Lrig-1 present on the surface of regulatory T cells, tyrosine phosphorylation of Stat3 protein present in stimulated regulatory T cells was performed by phosphotyrosine immunoblot ( tyrosine phosphorylation) was analyzed, and the results are shown in FIG. 10 .
  • the Lrig-1 protein-specific monoclonal antibodies (GTC210-01, GTC210-02, GTC210-03 and GTC210-04) according to the present invention showed the same level of phosphorylation of Stat3 as that of Th17 cells. It was confirmed that an increase in On the other hand, Lrig-1 protein-specific monoclonal antibodies (GTC110-01, GTC110-02, GTC110-03 and GTC110-04) according to the present invention continuously maintain Stat3 phosphorylation at the same level as iTreg cells and reduction was observed.
  • autoimmune diseases of Preparation Examples 1 to 4 which are monoclonal antibodies according to the present invention
  • CD45RB (high) cells were adoptively transplanted into RAG-1 -/- mice to autoimmune diseases.
  • IBD inflammatory bowel disease
  • the antibodies of Preparation Examples 1 to 4 were intraperitoneally injected in an amount of 200 ⁇ g/mouse, and then the treatment effect of autoimmune disease was analyzed, and the results are shown in FIG. 11 shown in
  • the Lrig-1 protein-specific monoclonal antibodies (GTC210-01, GTC210-02, GTC210-03 and GTC210-04) according to the present invention significantly reduce body weight in inflammatory bowel disease-induced mice. inhibition could be confirmed.
  • the Lrig-1 protein-specific monoclonal antibody according to the present invention can treat autoimmune diseases, graft-versus-host diseases, organ transplant rejection, asthma, It can be seen that immune-related diseases such as atopy or acute or chronic inflammatory diseases can be effectively prevented, improved or treated.
  • mice were subcutaneously injected with MOG peptide and intraperitoneally injected with Mycobacterium tuberculosis toxin.
  • MOG peptide intraperitoneally injected with Mycobacterium tuberculosis toxin.
  • another injection of Mycobacterium tuberculosis toxin was given to boost the immune system of the mice.
  • mice were administered the GTC210-01 antibody prepared in Preparation Example 1, and anti-IL-17a antibody was administered as a positive control.
  • mice is immobile, but one forelimb is responsive to stimulation
  • mice were subcutaneously injected with MOG peptide and intraperitoneally injected with Mycobacterium tuberculosis toxin.
  • MOG peptide intraperitoneally injected with Mycobacterium tuberculosis toxin.
  • another injection of Mycobacterium tuberculosis toxin was given to boost the immune system of the mice.
  • mice On the 2nd, 4th, and 6th day, it was administered to mice at a concentration of 5mg/kg of the GTC210-03 antibody prepared in Preparation Example 3.
  • mice is immobile, but one forelimb is responsive to stimulation
  • Roxol Fast Blue was also put in xylene to deparaffinize, and then xylene was removed using 100% alcohol, followed by Roxol Fast Blue for the nerve tissue of the mouse. After staining with blue (Luxol Fast Blue) at 60° C. for 16 to 24 hours, followed by differential discoloration (differentiator), the results are shown in FIG. 15 .
  • an immune-related disease that is, an autoimmune disease
  • an experimental autoimmune encephalomyelitis (EAE) mouse disease model was used.
  • Splenocytes of mice treated with the GTC210-03 antibody of Preparation Example 3 were isolated and CD4 T cells were reactivated.
  • the mouse since the mouse has already been exposed to the antigen in the body, when stimulated again outside the body, stronger activity occurs, and the differentiated CD4 T cells secrete their own representative cytokines.
  • cells were obtained on the 10th day after disease induction and reactivated for 48 hours with 40 ⁇ g/ml of MOG peptide. Then, the amount of cytokines present in the culture medium was confirmed by ELISA.
  • helper T cell 1 CD4+ T-bet+
  • helper T cell 17 CD4+ RoR ⁇ t+
  • regulatory T cells CD4+ Foxp3+
  • various inflammatory cytokines IFN-gamma, IL-17A
  • anti-inflammatory cytokines IL-10 increased expression.
  • the GTC210-03 antibody of Preparation Example 3 that specifically binds to the epitope according to the present invention when administered, it suppresses the expression of “inflammatory” cytokines as well as the expression of IL-10, an “anti-inflammatory” cytokine It was confirmed that the inflammatory response was eventually suppressed by increasing
  • mice 7-month-old Alzheimer's-induced 5xFAD mice (known to exhibit amyloid deposits, gliosis, and progressive neuronal loss with cognitive and motor deficits) were injected with GTC110-04 mouse antibody and GTC210-01 antibody at an amount of 10 mpk each. Injected intravenously for 1 month.
  • GTC110-04 mouse antibody As a positive control, 8-month-old Alzheimer's mice were subcutaneously injected with 100 ⁇ g of glatiramer acetate (GA, Copaxone®) for 3 weeks.
  • GA glatiramer acetate
  • mice of the group were treated with a Y-maze test, a Novel Object Recognition test, and a Water maze test.
  • a maze frame made by arranging the same three arms with a length of 40 cm (height of the wall 15 cm) at an angle of 120 degrees was used.
  • This experiment was a behavioral experiment using rodents' instinctive search habits, and it was a method that focused on the high possibility of exploring new areas.
  • the arm that was searched just before was memorized, and the higher the level of memory was displayed, the more they did not enter the same arm.
  • a search time of 8 minutes was provided per subject, and the final result was expressed as a spontaneous alteration (%) value in FIG. 17 .
  • the spontaneous alteration (%) value was calculated by Equation 1 below.
  • the behavioral pattern analysis was performed using SMART VIDEO TRACKING Software (Panlab, USA), and the results are shown in FIG. 17 .
  • Alzheimer's-induced mice were treated with the antibodies according to the present invention (GTC210-01 and GTC110-04 antibodies). When administered, it was confirmed that the spontaneous shift value was similar to that of the normal control group.
  • the normal control group (G1) was 0.51 ⁇ 0.06
  • the G2 group (vehicle) was measured as 0.42 ⁇ 0.04
  • the Alzheimer's induced group (G2) was measured as a normal control group.
  • the preference value was observed to be lower.
  • the antibodies according to the present invention GTC210-01 and GTC110-04 antibodies
  • a probe test was conducted on the 6th day after swimming practice, and as spatial perception ability, the value calculated as a percentage of the total swimming time of the time spent in the quadrant where the platform was located (Percentage of time in SW area, %) , The time taken to reach the position where the platform was (Latency to target, sec) was measured, and the results are shown in FIGS. 19 and 20.
  • analysis was performed using SMART VIDEO TRACKING Software (Panlab, USA). The criterion to exclude from the interpretation of the results was set as an individual turning only a specific part without searching through swimming.
  • the time spent in the quadrant where the platform was located was calculated as a percentage of the total swimming time (Percentage of time in SW area, %) was significantly increased, and the time taken to reach the position where the platform was located (Latency to target, sec) significantly decreased to a level similar to that of the normal control group.
  • IAA Iodoacetamide
  • Syringe filter 0.2um (Sartorius stedim, 16534)
  • the ratio of antigen and antibody was set to 2:1, and antigen samples were prepared. Then, DEPC was added so as not to exceed 1%, followed by reaction at 37 ° C. for 1 minute, and imidazole was added to complete the reaction. Then, after performing the precipitation reaction by adding acetone, the supernatant was removed by centrifugation, and the precipitate was well released using 0.1% PM. Trypsin, chymotrypsin, or Asp-N/Lys-C was added to the precipitate released in this manner to undergo degradation, and then sugar chains attached to proteins were removed using PNGase-F. Finally, disulfide bonds between cysteines of proteins were cleaved using DTT and IAA, and LC-MS and MS2 analysis were performed.
  • the ratio of antigen and antibody was set to 2:1, and two antigen samples were prepared. Then, the cross linker (CSBU) was sufficiently dissolved in DMSO. Thereafter, the ratio of the sample and DSG was added to be 1:100, and only DMSO was added to the other, followed by reaction at 25 °C for 1 hour. After the reaction was completed, the protein was decomposed using trypsin or chymotrypsin, and sugar chains attached to the protein were removed using PNGase-F. Finally, disulfide bonds between cysteines of proteins were cleaved using DTT and IAA, and LC-MS and MS2 analysis were performed.
  • CSBU cross linker
  • Step total time (min) Flow rate ( ⁇ L/min) A(%) B(%) 0 0.00 100.00 95.0 5.0
  • Control group huTregL1-his (Ag, Control) and test group huTregL1-his/E7-mlgG2a (Ag+Ab, Test) samples were treated with DEPC and then treated with trypsin, chymotrypsin or Asp-N/Lys-C to digest Each sample was analyzed three times repeatedly (see FIGS. 21 to 23).
  • the MS analysis results of the control group (Control 1) and the test group (Test 1) shown in the above analysis results were matched with the sequence of the control group (huTregL1-his) using the BioPhama finder program, and the labeled peptides were compared.
  • sequences E62-L101, D111-L134, and Q542-Y553 were 13.2%, 15.3%, and 14.7%, respectively, with a labeling decrease (%) of 10% or more.
  • the E62-L101 sequence the E62-K92 part (AL) showed 65.9%, L65-F87 part (Y) 48.7%, and the L88-L101 part showed labeling (%) of 88.6%. From this result, it can be considered that the L88-L101 portion with labeling (%) of 50% or more among the E62-L101 sequences is exposed to the outside.
  • the chymotrypsin-treated group showed a 15.3% labeling decrease (%), but it was confirmed that the control RSD was 62.4%.
  • the Q542-Y553 sequence also showed a 14.7% labeling decrease (%) in the chymotrypsin-treated group, and the labeling (%) of the control antigen was 50% or more, which was judged to be an externally exposed portion in terms of protein structure.
  • test group (huTregL1-his/E7-mlgG2a) mixture sample was prepared, the test group sample was treated with a cross-linker, disuccinimidyl dibutyric urea (DSBU), and the test group was treated with a protease (chymotrypsin or trypsin). Samples were disaggregated (FIGS. 27 and 28). Then, peptide sequences in which peptides digested with chymotrypsin and trypsin were not detected in the test tube (including 2% or less) were identified in comparison with the control group.
  • DSBU disuccinimidyl dibutyric urea
  • the peptide sequences not detected in the test group samples were combined and determined as the epitope site.
  • the peptide ratio (T%) of the same sequence detected in the test group sample compared to the control group was confirmed to be 2% or less in both chymotrypsin or trypsin. .
  • T158-F174 and G442-K476 were decreased in Chymotrypsin (Y) and Trypsin (T), respectively.
  • S362-K365 of the Trypsin (T) treated group was the only sequence where T% was not detected (0%).
  • T% was not detected (0%).
  • the epitope was predicted by synthesizing the previous results. Specifically, two sequences of huTregL1-his (Ag), including E62-L101 and G452-K476, were identified as identical positions in CL-MS and XL-MS analysis results. Among the L62-L101, the L88-K92 part showed a labeling decrease (%) in the two protease treatment groups (Chymotrypsin, Asp-N/Lys-C) as a result of CL-MS, and in the XL-MS analysis, the two protease treatment groups ( Chymotrypsin, Trypsin) were significantly reduced.
  • the S95-L101 portion In the case of the S95-L101 portion, it was an epitope overlappingly identified in chymotrypsin and trypsin in XL-MS. In addition, as a result of CL-MS, the L88-L101 portion with labeling (%) of 50% or more was exposed to the outside. In particular, in the case of L88-L101, the amino acid sequence in the sequence was composed of hydrophilic amino acids (Q89-S95) consecutively.
  • the antibody or antigen-binding fragment specifically binding to the epitope of Lrig-1 protein according to the present invention inhibits the function of regulatory T cells by specifically binding to the epitope of the present invention present on regulatory T cells, and By maintaining or increasing the activity of T cells, it can be used very efficiently for the prevention, improvement or treatment of various diseases.

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Abstract

La présente invention concerne un épitope d'une protéine à domaines riches en leucine et de type immunoglobuline 1 (Lrig1), qui est un antigène présent sur la surface de lymphocytes T régulateurs, ainsi qu'un anticorps ou un fragment de liaison à l'antigène se liant spécifiquement à celui-ci.
PCT/KR2022/010262 2021-07-16 2022-07-14 Épitope d'antigène de surface de lymphocyte t régulateur, et anticorps se liant de manière spécifique à celui-ci WO2023287212A1 (fr)

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CN202280050296.0A CN117716238A (zh) 2021-07-16 2022-07-14 调节性t细胞表面抗原的表位及其特异性结合的抗体
EP22842474.3A EP4375670A1 (fr) 2021-07-16 2022-07-14 Épitope d'antigène de surface de lymphocyte t régulateur, et anticorps se liant de manière spécifique à celui-ci
US18/413,322 US20240141036A1 (en) 2021-07-16 2024-01-16 Epitope of Regulatory T Cell Surface Antigen, and Antibody Specifically Binding Thereto

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4554101A (en) 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
KR20180117066A (ko) * 2017-04-18 2018-10-26 주식회사 굳티셀 Lrig-1 단백질에 특이적인 결합 분자 및 이의 용도
KR20190027808A (ko) * 2019-03-08 2019-03-15 주식회사 굳티셀 조절자 Ｔ 세포에 특이적으로 존재하는 새로운 표면단백질 Ｌｒｉｇ-1의 용도
KR20200112745A (ko) * 2019-03-20 2020-10-05 주식회사 굳티셀 뇌신경계 질환의 예방 또는 치료용 조성물
KR20210056280A (ko) * 2019-11-08 2021-05-18 주식회사 굳티셀 조절 t 세포 표면 항원의 에피토프 및 이에 특이적으로 결합하는 항체
KR20210075117A (ko) * 2018-10-05 2021-06-22 세인트 안나 킨더크렙스포르슝 키메라 항원 수용체(car) 그룹

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4554101A (en) 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
KR20180117066A (ko) * 2017-04-18 2018-10-26 주식회사 굳티셀 Lrig-1 단백질에 특이적인 결합 분자 및 이의 용도
KR20210075117A (ko) * 2018-10-05 2021-06-22 세인트 안나 킨더크렙스포르슝 키메라 항원 수용체(car) 그룹
KR20190027808A (ko) * 2019-03-08 2019-03-15 주식회사 굳티셀 조절자 Ｔ 세포에 특이적으로 존재하는 새로운 표면단백질 Ｌｒｉｇ-1의 용도
KR20200112745A (ko) * 2019-03-20 2020-10-05 주식회사 굳티셀 뇌신경계 질환의 예방 또는 치료용 조성물
KR20210056280A (ko) * 2019-11-08 2021-05-18 주식회사 굳티셀 조절 t 세포 표면 항원의 에피토프 및 이에 특이적으로 결합하는 항체

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